Spermine (Spm) and its aromatic derivative BD23 modify the morphology of Pyrus communis pollen tubes by inducing ROS scavenging

Pollen tube growth is a rapid process restricted to the only tip region. Many factors cooperate to allow this apical growth, creating an intricate signalling network. The continuous rebuilding of the cell wall and apical migration of the cytoplasm sustained by cytoskeleton re-organisation are the most important driving forces needed for growth1, but many other factors are involved in this process, among which polyamines (PAs), that are essential during pollen tube emergence2 and ROS, that support the apical growth, at physiological concentration3. We investigated the effect of the natural PA spermine (Spm) and BD23, a synthetic aromatic derivative of Spm on the apical growth of Pyrus communis pollen tube and observed that both Spm inhibited the growth from 10 M onwards. Thanks to a FITC-labelled Spm, we were able to observe, that PAs enter through the pollen tube tip, then diffuse in the sub-apical region. The same region underwent drastic morphological changes, showing loss of polarity and enlarged tip when Spm and BD23 were supplied at 100 M or higher. The effects of PAs concern, at least in part, their ability to modulate ROS concentration. In fact the activity of NAD(P)H-oxidase (NOX), catalase (CAT) and superoxide dismutase (SOD) was drastically affected by PAs supplementation. Moreover, also the tip-focused Ca2+-gradient was suddenly altered. Interestingly, the actin cytoskeleton was affected by the treatment with 100 M of both the PAs causing its re-organization in thicker bundles that followed the swelling of the apex. Intriguing, the cell wall was enriched in both callose and cellulose exactly in the region where the swelling of the tip begins, probably to contrast the excess of ductility of the cell wall after treatment with PAs 100 M. The viability of pollen was slightly affected by 100 M treatment, and the degradation of nuclear DNA, was completely inhibited when pollen had been pre-treated with the caspase-3 inhibitor I peptide, Ac-DEVD-CHO (DEVD). Different was the scenario when the PAs were supplemented at 500 M, concentration that is far away from the physiological one. In this case, the effects on the pollen were more drastic, with a rapid drop of cell viability, actin depolymerisation, stimulation of DNA-laddering after 30 minutes incubation and the complete degradation of both vegetative and generative nuclei; these effects were only in part inhibited by the pre-treatment with DEVD. Thus, the present data may open new research avenues to understand how the diameter of the pollen tube is regulated and which role PAs could play in the puzzling process of apical growth.